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Improved Immunoassay Strategies for The Determination of Mycotoxins and Steroid Hormones

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Title Improved Immunoassay Strategies for The Determination of Mycotoxins and Steroid Hormones
 
Creator Acharya, Debopam
 
Subject Drug Development/Diagnostics & Biotechnology
 
Description Immunoassay techniques are specific, sensitive, cost-effective, robust and amenable to on-site monitoring. Depending on format, the assays fall into two main categories, non-competitive and competitive immunoassays. Noncompetitive assays, mostly used for large molecules and proteins, are based on two different antibodies with ability to bind simultaneously to two independent epitopes on the analyte. Analysis of low-molecular-mass analytes, such as mycotoxins, steroid hormones etc., not large enough to bind two antibodies simultaneously, require a competitive immunoassay format. In this format, the analyte competes with a labelled (or immobilized) analyte analogue (hapten), and the measured signal is inversely proportional to the analyte concentration. Mathematical modeling of immunoassay performance shows that competitive assays are inferior to noncompetitive ones in terms of sensitivity, precision, kinetics, and working range. For this reason, several attempts have been made to implement small molecule noncompetitive assays but till date only a few examples have been reported. Further, immunoassays in general have the limited capability to analyze multiple analytes simultaneously.
During the present investigation, work has been carried out to overcome some of the above disadvantages. The investigations are summarized below:
Simultaneous assay of aflatoxin B1 (AFB1) and ochratoxin A (OTA)
A membrane-based competitive immunoassay has been developed for the estimation of structurally diverse mycotoxins, AFB1 and OTA in chili samples. The assay was carried out over an analytical device, which consists of nitrocellulose membrane strips attached at one end of a polyethylene card with a filter paper placed below the membrane over the polyethylene card. This improved device differs from the earlier reported ones in two aspects. (1) The attachment of pre-wetted absorbent body below the membrane surface was not required, and (2) optimal water content can be maintained in the absorbent body simply by shaking the device 2-3 times. The membranes are marked into several zones, and 1 μL of each antibody (anti-AFB1 and anti-OTA) in duplicate are immobilized uniformly, which generate four antibody spots in each zone. During an assay, 25 μL of standards or samples are applied on antibody spotted zones. After washing and removal of excess water from the filter paper, 25 μL of a mixture of enzyme conjugate (AFB1-HRP + OTA-HRP) is added. Visualization may be accomplished by the addition of a mixed substrate solution (4-Chloro-1-naphthol + 3,3'-Diaminobenzidine). The combined detection of AFB1 and OTA is more economical in respect of time, work and material than two individual assays. Chili sample extracts containing high amount (40%) of methanol were directly analysed after 2-fold dilution with assay buffer. The results can be analysed either by visual comparison of the colour intensity of a sample spot with those of reference standards or more precisely by densitometry. The limits of quantitation for AFB1 and OTA were found to be 2 and 10 μg kg-1 respectively. Average recoveries for spiked samples of AFB1 were found within 92 - 120% whilst for OTA it was observed within 93 - 110%. The observed amounts of AFB1 and OTA present in chili samples correlated well with those determined by individual ELISA. Thus, the method is well suited for simultaneous determination of AFB1 and OTA in chili samples to meet the regulatory requirements. Although the investigation has been restricted to two analytes, it is obvious that this approach may be exploited for simultaneous screening of more than two analytes.
 
Date 2008
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/837/1/FINAL_THESIS_FOR_LIBRARY.pdf
Acharya, Debopam (2008) Improved Immunoassay Strategies for The Determination of Mycotoxins and Steroid Hormones. PhD thesis, Jadavpur University.
 
Relation http://www.eprints.iicb.res.in/837/